Field of the Invention
The present invention relates generally to the fields of molecular biology, and more specifically to tau protein, and protease-active fragments and variants thereof. In particular embodiments, truncated tau fragments and tau protease variants are provided that retain substantial serine protease.
Description of Related Art
Alzheimer's Disease
There is a large and rapidly growing unmet need for disease modifying drugs for Alzheimer's disease. Currently more than 30 million people suffer from AD worldwide and this number doubles about every 20 years. In the US, it is estimated that there are 5.4 million AD sufferers. AD affects 1 out of 4 people over age 75 and 1 out of 3 people over 80. Payments for care in 2012 are estimated to exceed $200 billion (2012 Alzheimer's disease Facts and Figures, Alzheimer's Association). Presently, only 5 mildly effective AD symptom-treating drugs exist, but none that treat the underlying neurodegenerative processes. FDA approved medications provide limited symptomatic relief, but do not halt, slow or reverse disease progression. It is estimated that the 2009 market for anti-Alzheimer's drugs was approximately $4.3 billion and is expected to increase to over $14 billion by the end of the decade based on the introduction of disease modifying drugs (DMDs), which are expected to fuel >50% of the market growth.
The symptoms of AD manifest slowly and the first symptom may only be mild forgetfulness. In this stage, individuals may forget recent events, activities, the names of familiar people or things and may not be able to solve simple math problems. As the disease progresses into moderate stages of AD, symptoms are more easily noticed and become serious enough to cause people with AD or their family members to seek medical help. Moderate-stage symptoms of AD include forgetting how to do simple tasks such as grooming, and problems develop with speaking, understanding, reading, or writing. Severe stage AD patients may become anxious or aggressive, may wander away from home and ultimately need total care.
The classical hallmarks of AD are inter-neuronal plaques consisting of precipitates or aggregates of amyloid β protein (Aβ), and intra-neuronal neurofibrillary tangles (NFTs) consisting of precipitates or aggregates of tau protein. The amyloid cascade hypothesis has been widely accepted as the pathological pathway of AD, that Aβ drives AD pathogenesis and secondarily induces the formation of abnormal tau protein. Genetic evidence suggests that that mutations leading to increased accumulation of Aβ aggregates leads to familial AD. However, there are a number of weaknesses in the Aβ cascade hypothesis in that it does not address the importance of other pathways that can cause neurodegeneration (Seabrook et al., 2007). The accumulation and distribution of NFTs in the brains of AD patients is highly correlated with disease progression and can be used to stage AD by post-mortem brain histopathology. A recent study was conducted in which two thousand three hundred and thirty two non-selected brains from 1- to 100-year-old individuals were examined for abnormal tau and for the detection of Aβ. This study showed that AD-related tauopathy begins in the early decades of life in the lower brainstem and before the occurrence of plaques contradicting the amyloid cascade hypothesis (Braak et al., 2011). Furthermore, there have been a number of late stage clinical failures that call into question the understanding of the molecular mechanism of AD pathology (see, e.g., Table 1.).
TABLE 1LATE-STAGE FAILURES OF DRUGS TARGETING AβCompoundMode of ActionCompanyStatusTramiprosateβ-amyloidNeurochemFailed to meet(Alzhemed ™)antagonistclinical endpointin Phase IIIR-flurbiprofenReduces levelsMyriadFailed to meet(Flurizan ™)of Aβ42Geneticsclinical endpointin Phase IIISemagacestatγ-SecretaseEli LillyFailed to meet(LY450139inhibitorclinical endpointdihydrate)in Phase IIIBapineuzumabHumanizedWyeth/Elan,Subjects weremAB specificPfizer/J&Jstratified accordingfor the endsto APOE genotypeof Aβin order to meetPhase II endpointCausal Role for Tau in Neurodegenerative Diseases
That tau dysfunction is sufficient for neurodegeneration and dementia, even in the absence of other disease processes comes from direct evidence that mutations in the gene for tau MAPT cause frontotemporal dementia with Parkinsonism (FTDP) linked to chromosome-17 (FTDP-17). The 32 different mutations found in the study of over 100 families can be grouped into categories influencing splicing of the primary transcript and causing changes in amino acid sequence of tau. Most missense mutations are located in the assembly domain and generally reduce the affinity of tau to MTs. Several of these mutations promote aggregation of tau in vitro and in vivo such as P301L and P301S. Mutations in the stem-loop structure at the border of exon 10 and the following intron alter splicing causing aberrations in the ratio of 4R to 3R isoforms demonstrating that maintenance of the proper ratio of tau isoforms is necessary to prevent neurodegeneration and dementia (Goedert and Jakes, 2005). Recent work has shown that tau is a key mediator or enabler of both Aβ- and apoE4-dependent pathogenesis (reviewed in Morris et al., 2011; Huang and Mucke, 2012).
Extracellular Tau in Disease Progression
The role of extracellular tau in neurotoxicity is a relatively new but important concept in the field. There are a number of contributing findings that implicate extracellular tau in AD. Tau pathology spreads contiguously throughout the brain from early to late stage disease suggesting an “infectious” model of disease progression (Schonheit et al., 2004). This notion is supported by a recent report (Frost et al., 2009) that extracellular tau aggregates can propagate tau misfolding from outside to the inside of a cell. Additional backing for this concept comes from a recent report showing that injection of brain extract from a transgenic mouse with aggregated mutant human tau into the brain of transgenic mice with normal human tau transmits tau pathology and induces its spread throughout the brain (Clavaguera et al., 2009). Recently two transgenic models were independently developed that expressed pathological tau P301L in mouse entorhinal cortex (Liu et al., 2012; de Calignon et al., 2012). The published results demonstrated that the pathology spread to adjacent regions of the hippocampus consistent with the model that tau pathology can spread from diseased to healthy neurons. Induction of low levels of pro-aggregation human tau in transgenic mice results in the formation of tau aggregates and tangles composed of both human and normal murine tau (co-aggregation) providing evidence for the “infectious” model by transmission of pathological tau characteristics to normal host tau (Mocanu et al., 2008). A receptor-mediated mechanism for the spread of tau pathology by extracellular tau has been described based on work with cultured neurons (Gomez-Ramos et al., 2006; 2008; 2009). Levels of tau rise in CSF in AD, whereas Aβ levels decrease (Shaw et al., 2009).
Tau Oligomers: A Target for Therapeutic Development
NFTs have been implicated in mediating neurodegeneration in AD and tauopathies as it correlates well with cognitive deficits and neuron loss (Arriagada et al., 1992; Bancher, 1993; Guillozet et al., 2003; Iqbal et al., 2009). However, the study of animal models of tauopathy has shown that memory impairment and neuron loss is dissociated from accumulation of NFT (Brunden et al., 2008). Strong support for this contention came from the analysis of transgenic mice rTg4510 that express tau P301L in the forebrain under control of a tetracycline-regulated promoter. These mice developed memory impairment, neuron loss and NFT when the construct was expressed. However, suppression of expression caused improvement in memory and reduction in neuron loss even as NFTs continued to accumulate clearly demonstrating that pretangle tau species were responsible for the neurodegenerative phenotype (Santacruz et al., 2005). Additionally, there was regional dissociation of neuron loss and NFT pathology (Spires et al., 2006). This mouse model was also used to show that soluble tau, but not tangles, contributed to impairment of hippocampal function (Fox et al., 2011). Transgenic mice expressing a human mutant tau P301S construct prone to aggregation developed hippocampal synapse loss and dysfunction, as well as, microglial activation months before the accumulation of filamentous tau inclusions (Yoshiyama et al., 2007). Similarly, a transgenic mouse model expressing human tau protein with two mutations found in FTDP-17 (P301S and G272V) exhibited axonopathy before tangle formation (Leroy et al., 2007). The triple transgenic AD mouse model accumulating both tau and Aβ pathology was used to study the effects of immuno-reduction of tau and Aβ. Antibodies against both proteins were needed to improve learning and memory behavior in these mice. Soluble tau, but not NFT, was reduced by the treatment (Oddo et al., 2006).
A study of normal and AD CSF specimens using a tau oligomer-specific antibody showed AD-specific accumulation of tau oligomers early in disease (Lasagna-Reeves et al., 2012). Furthermore, tau oligomers caused impairment of memory and induced synaptic and mitochondrial dysfunction in mice (Lasagna-Reeves et al., 2011). However, the mechanism by which tau oligomers cause these neurodegenerative effects has not been established.
Proteases Cutting Tau
Tau cleavage has been shown to play an important role in tau aggregation and neurodegeneration (recently reviewed in Wang el al., 2010; Hanger and Wray, 2010). Tau truncation leads to the formation of aggregation-prone fragments leading to the formation of toxic aggregates and leads to the formation of toxic fragments which do not aggregate. Thus, targeting the proteolysis of tau would be beneficial for the development of therapeutics for AD and related tauopathies. Tau is a substrate for multiple proteases and because of its natively unfolded conformation it is very susceptible to proteolysis. Tau can be cut by trypsin and chymotrypsin in addition to endogenous proteases such as caspases, and calpain and puromycin-sensitive aminopetidase. The protesome, which degrades misfolded proteins, also degrades tau but is inhibited when bound to filaments of tau. There are also unknown proteases that generate fragments of tau early in AD.
The Role of Tau in Alzheimer's Disease
The classical hallmarks of AD are inter-neuronal plaques consisting of precipitates or aggregates of amyloid beta protein (Aβ), and intra-neuronal neurofibrillary tangles (NFTs) of tau protein. Tau protein promotes microtubule assembly and stability and is critical for the function of axons, whereas the normal function of Aβ is not fully understood. The amyloid cascade hypothesis has been widely accepted as the pathological pathway of AD. It holds that the generation of Aβ and accumulation of Aβ aggregates in the brain initiate the disease process. It is supported by genetic evidence that mutations leading to increased accumulation of Aβ aggregates leads to familial AD. However, there are a number of weaknesses in the Aβ cascade hypothesis in that it does not address the importance of other pathways that can cause neurodegeneration (Seabrook et al., 2007). The accumulation and distribution of NFTs in the brains of AD patients is highly correlated with disease progression and can be used to stage AD by post-mortem brain histopathology, whereas there is poor correlation between AD and the accumulation of neuritic plaques composed of β-amyloid. This has been used to challenge the amyloid hypothesis (Josephs et al., 2008). Lackluster results for Aβ-directed therapeutics in late stage clinical trials has increased interest in exploring alternative targets for drug discovery such as tau (Iqbal et al., 2009).
Deficiencies in the Prior Art
Unfortunately, no cure is yet available for AD. Today, medication therapy focuses on controlling the symptoms of AD and its various stages. For example, mild to moderate AD is often treated with cholinesterase inhibitors such as donepezil (ARICEPT®, Eisai Co., Ltd/Pfizer, Inc.), rivastigmine (EXELON®, Novartis AG/Sandoz AG), galantamine (RAZADYNE®, Johnson & Johnson), [and to a lesser extent, tacrine (COGNEX®), Warner-Lambert Co.], while moderate to severe AD is often treated with donepezil (ARICEPT®) or N-methyl D-aspartate antagonists such as memantine (NAMENDA®, Forest Laboratories, Inc.), or a combination thereof. Although these medications may help delay or prevent AD symptoms from becoming worse for a limited period of time, there is no clear evidence that these medications have any effect on the underlying progression of the disease itself.
While extensive research in the past decade has identified possible biomarkers for AD, there is still an urgent need for composition and methods that are specifically useful in diagnosing, treating, preventing, and monitoring the progress of AD in at-risk or affected individuals. New compositions and methods are also needed to serve as drug targets for the identification, synthesis, and/or adaptation of existing chemical compounds for use in the treatment of AD and its symptoms. Furthermore, there is also a need for development of new classes of drugs for treatment of the disease, including, for example, immunotherapeutic agents and next-generation therapeutics.